Grape juice, must and other fruit juice contain a varying amount of L-malic acid and L-malate, the amount typically being in the range of 1 to 10 g/l. The amount of malic acid and malate depends largely on the climatic conditions prevailing in the viticultural region. Hence, wines produced in colder areas tend to have a relatively higher acid content, since the malic acid is not degraded during the normal alcoholic fermentation. From a taste and flavor point of view, malic acid is considered undesirable in most red wines and in several types of róse wines, white wines or sparkling wines.
However, the content of malic acid and malate in a wine may be reduced by a so-called malolactic fermentation (MLF) of the wine which fermentation results from the metabolic activity of various lactic acid bacteria, including species belonging to the genera of Lactobacillus, Pediococcus and Leuconostoc. Such bacteria may be present in must and wine as part of the indigenous microbial flora hereof, or they may have been added as a bacterial starter culture. Typically, the MLF is associated with malolactic bacterial growth and catabolic processes during which the wine acidity is reduced. The catabolic phase is usually entered when the malolactically active bacteria during the growth phase has reached a population density of about 106 colony forming units (CFU) per ml. The microbial malolactic deacidification results from the decarboxylation of the dicarboxylic acid, L-malic acid to the monocarboxylic acid, L-lactic acid. As a result of this malolactic fermentation, the acidity of the wine decreases and the pH increases, resulting in a wine with a softer palate relative to that of the wine before the malolactic fermentation. Following a successful malolactic fermentation in wine, no further microbial growth will normally occur and hence, the wine is considered to be microbiologically stable.
The malolactic fermentation may occur spontaneously in the wine as a result of the growth of an indigenous flora of malolactically active lactic acid bacteria originating from the vines and grape skins and also often surviving from one season to the next on winery equipment, especially wooden casks or other equipment made of wood. When occurring in this fashion, malolactic fermentation is often delayed and may take place several months after the alcoholic fermentation. The initial number of bacteria is often quite small and the environment of the wine is frequently rather hostile to the growth of these bacteria due to the content of ethanol and sulphur dioxide in the wine, as well as its low pH and low nutrient concentration. The extended lag phase of the malolactic bacteria during which the wine is biologically unstable may result in the growth of bacteria producing volatile acidity and hence spoilage of the wine. Apart from this, certain indigenous malolactic bacteria spontaneously growing in the wine may produce certain compounds, e.g. biogenic amines that are believed to give rise to health problems.
In the traditional winery industry where the spontaneously occurring malolactic fermentation is being relied upon, it is common practice to stimulate malolactic fermentation by reducing the amount of added sulphur when determined as sulphur dioxide, to below 50 mg per l, delaying the removal of the lees, increasing the temperature of the wine to above 20° C., or ensuring a pH of more than 3.4. These measures, however, may also favor the growth of undesired microorganisms in the wine such as Acetobacter species, thus increasing the likelihood of wine spoilage, and this approach therefore requires extremely careful supervision of the decarboxylation process. Even if precautions are taken to enhance spontaneous MLF, this process is still difficult, not to say impossible, to control, and its occurrence has become even more unpredictable as winery hygiene has improved e.g. as a result of the replacement of wooden casks with stainless steel tanks. Such hygienic measures serve to ensure a uniform quality of the wine and reduce the risk of spoilage. However, they also reduce the chance of spontaneous conversion of malic acid taking place in the wine.
For this reason, and because the winemaker will often prefer to exercise a greater degree of control over the malolactic fermentation process it has become increasingly common practice in the winery industry to add a starter culture of malolactically active bacteria to the wine after the alcoholic fermentation. Presently, such a postfermentation malolactic fermentation in wine may be induced in different ways.
Currently, a widely practiced method of inducing MLF is to seed a wine with a small proportion of another wine already undergoing MLF and thus containing a high number of viable malolactically active bacteria. The culture in the seed wine is then already well adapted to wine conditions and will usually be capable of completing the malolactic fermentation in the seeded wine. However, this method of inoculation is rather tedious and not completely controllable. Thus, the method requires that a concentrated “mother culture” of the bacteria is propagated for an extended period of time, such as about two months, in diluted wine or grape juice, optionally after rehydration and/or adaptation of the mother culture in e.g. a grape fruit juice-containing medium which is then used to inoculate the wine to be used as a bulk starter culture in the form of seed wine. Typically, the wine is inoculated with the seed wine at a rate of 1 to 10 vol %, and accordingly, this method requires substantial investments in propagation equipment and adequately trained staff. In addition, it is difficult to control the propagation process and hence serious timing problems may occur. Should both red and white wine starter cultures be required these problems are doubled.
Relative to the above practice of using a seed wine, a post-fermentation inoculation of wine with concentrated freeze-dried or frozen starter cultures of malolactically active lactic acid bacteria implies considerable savings in time and labor by substantially reducing the amount of scale-up work required. Such concentrated starter cultures are now commercially available and their use is being increasingly accepted in the wine industry, although their malolactic effect is not completely reliable. These culture compositions may be concentrated to contain a number of colony forming units which, when determined in a non-inhibitory medium is in the range of 109 to 1011 per g of culture.
However, the use of known, commercially available malolactically active culture compositions contain bacteria which are susceptible to the conditions (low pH, content of SO2, content of ethanol) prevailing in the fermented wine and accordingly, their malolactic efficiency requires that they become adapted to the hostile conditions in the wine by carrying out a thawing and rehydration step (if freeze-dried), a thawing step (if frozen) and an activation step prior to inoculation in the wine. This requirement is assumingly due to the well-known phenomenon that a freeze-dried or frozen lactic acid bacterial culture composition, even if it includes additives protecting the bacteria against cell damages, will as a result of the freezing and/or freeze-drying process have an increased susceptibility to low pH, SO2, ethanol and low temperatures as compared to a freshly grown culture of the same species.
If not rehydrated and activated as described above, the survival rate of the known commercial malolactic compositions on direct inoculation into wine will typically be in the range of 0.01 to 1% or even lower. Furthermore, the initially surviving non-adapted bacteria may gradually lose their viability in the wine.
Typically, this required adaptation comprises an initial thawing and/or rehydration process, the latter comprising dissolving the freeze-dried composition in water and adding various nutrients such as a sugar, vitamins, minerals or yeast extract and keeping the resulting solution at about 22° C. for about one hour. Subsequently, the thus rehydrated composition is subjected to an activation step, typically lasting 48 to 84 hours, in a medium which typically comprises grape juice or wine diluted with water, yeast extract, trace elements and vitamins. Normally, the number of CFUs does not increase during this activation period, on the contrary, the number may decrease. Even if the starter culture composition is adapted as described above, a varying proportion of adapted bacteria may lose viability as determined in a non-inhibitory medium when inoculated in wine. This loss of viability may be up till 90% of the added number of CFUs.
In EP-A1-0141878 is disclosed a method of reducing malic acid to lactic acid in wine by the introduction of high numbers of bacterial cells into the wine, which method comprises activating a concentrate of a bacterial culture in a nitrogen source-augmented fruit juice to form an activated mixture of bacteria containing at least about 105 CFUs per ml and introducing the activated mixture into wine or grape must and converting the malic acid to lactic acid. The activation conditions disclosed are an activation period of 48 hours at 24° C.
Krieger et al. (Food Biotechnol. 1990, 7, 484) have disclosed the application in wine of malolactically active strains of Leuconostoc oenos (Ln. oenos) and Lactobacillus spp. in the form of fresh or frozen concentrates using direct inoculation, i.e. without preceding activation, of at least 107 CFUs per ml which concentration is indicated as being necessary to start malolactic fermentation. However, the application of such a high concentration of malolactically active organisms is not commercially feasible in the wine industry due to the high cost of starter cultures. The necessity to apply at least 107 CFUs per ml as disclosed in order to start MLF may indicate that the survival rate of these organisms when applied directly to the wine is so low that a malolactically active concentration of the organisms is only achieved at the indicated inoculation level.
The present invention provides, compared to the known methods, a significantly improved method of inducing malolactic fermentation in wine or fruit juice whereby it has become possible to achieve an effective malolactic fermentation herein within a short period of time by inoculating wine or a fruit juice directly with a concentrated culture composition of malolactically active bacteria at an economically feasible concentration and accordingly, to avoid the tedious and costly processes of rehydration, activation, adaptation and/or expansion which are currently required with commercial malolactically active starter cultures.